ES2648788T3 - Method to assess a subject's risk of having cardiovascular disease - Google Patents

Abstract

A method for assessing a subject's risk of having a cardiovascular disease comprising the step of measuring the level of inhibition factor 1 (IF1) in a body fluid sample obtained from said subject in which the level of IF1 is negatively correlated with the risk of said subject of having a cardiovascular disease.

Description

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DESCRIPTION

Method to assess a subject's risk of having cardiovascular disease

5 Technical sector

The present invention relates to a method for assessing a subject's risk of having a cardiovascular disease.

State of the art

Despite important advances in the treatment of cardiovascular disease (CVD), it remains one of the leading causes of death in developed countries. Some large-scale prospective epidemiological studies provide significant evidence that low plasma cholesterol levels

15 with high density lipoprotein (HDL-C) is an important risk factor independent of CVD. Studies in genetically modified animal models and in subjects with rare disorders of HDL metabolism support a causal relationship between low levels of HDL and the development of atherosclerotic vascular disease. It is somewhat remarkable that a low level of HDL-C (generally considered by current treatment guidelines at <40 mg / dl or <1.0 mmol / l in men and <50 mg / dl or <1.3 mmol / l in women ) is still predictive of future cardiovascular risk, even if the LDL cholesterol concentration has reached low levels by statin treatment (Barter P, Gotto AM, LaRosa JC, et al. HDL cholesterol, very low levels of LDL cholesterol, and cardiovascular events N Engl J Med. Sep 27 2007; 357 (13): 1301-1310).

Although this atheroprotective activity of HDL is not widely accepted, the mechanism that supports this

25 effect continues to be debated. The best established mechanism by which HDLs protect against atherosclerosis is to stimulate the release of cholesterol from macrophages and the transport of cholesterol to the liver for excretion in bile and feces, a process called 'reverse cholesterol transport' (RCT ). In addition, this atheroprotection role of HDLs may also be related to their anti-inflammatory, anti-apoptotic, or endothelial protective properties, which are called pleiotropic effects (Calabresi L, Gomaraschi M, Franceschini G. Endothelial protection by high-density lipoproteins : from bench to bedside Arterioscler Thromb Vasc Biol. October 1, 2013; 23 (10): 1724-1731; von Eckardstein A, Hersberger M, Rohrer L. Current understanding of the metabolism and biological actions of HDL. Curr Opin Clin Nutr Metab Care. Mar 2005; 8 (2): 147-152). These pleiotropic protective effects of HDL lead to the concept that therapies aimed at improving plasma HDL-C levels would be antiatherogenic and protective against cardiovascular events.

35 However, HDLs are very heterogeneous, with subclasses that differ in density, size, load and protein composition, which have different functions and may differ in their functional antiatherogenic properties. In addition, several environmental factors, such as smoking, alcohol consumption and physical activity or inflammatory disease, are known to affect plasma HDL-C levels. Given the complexity of the HDL system, it has been theorized that a single measurement of HDL-C levels frequently fails to provide a reliable prediction of the biological activities of HDL to protect against CVD and, therefore, is often a poor indicator of the protection or risk of an individual subject. Therefore, new markers are needed that reflect the metabolic or vascular activities of the HDL particles in order to better assess the condition of the subject in terms of cardiovascular risk, or to assess the sensitivity of the subjects to novel treatments related to the HDL.

45 Rouslin et al (J Bioenerg Biomembr. August 1995; 27 (4): 459-66) shows the measurement of IF1 that involves sonicating mitochondria and incubating extracts containing IF1 with rat heart submitochondrial particles.

Di Pancrazio et al (Biochim Biophys Acta. November 4, 2004: 1659 (1): 52-62) shows the determination of IF1 by SDS gel electrophoresis, two-dimensional SDS PAGE and immunoblotting with an antibody directed against IF1 after extraction of IF1 of biopsies.

Navab et al (Circulation. June 29, 2004; 109 (25): 3215-20) report that D-4F (an mimetic peptide of oral apolipoprotein A-I) reduces atherosclerosis in mice.

55 Chapman et al (Eur Heart J. 2010 Jan; 31 (2): 149-64) evaluate the implication of cholesteryl ester transfer proteins (CETP) in the action of current liquid modulating agents with potential to increase HDL, and compare the mechanism of action with that of those developing pharmacological agents that directly inhibit cholesteryl ester transfer protein.

Mousa et al (Drugs Future. January 2010; 35 (1): 33-39) disclose several possibilities to modulate at the HDH level.

Spillman et al (Curr Pharm Des. May 2010; 16 (13): 1517-30) discloses different strategies to increase HDL levels. 65

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Besler et al (Curr Pharm Des. May 2010; 16 (13): 1480-93) discloses several agents that increase the level of HDL in cardiovascular diseases.

The PCT patent application published under number WO2006 / 029982 refers to the transfer proteins of the cholesteryl ester for the treatment of atherosclerosis.

Waksman et al. (J Am Coll Cardiol. June 15, 2010; 55 (24): 2727-35) discloses autologous serial infusions of selective cleared HDL plasma for treating patients with acute coronary syndrome.

Nissen et al (JAMA. November 5, 2003; 290 (17): 2292-300) evaluates the effects of recombinant ApoA-I Milano on coronary atherosclerosis in patients with acute coronary syndromes.

Object of the invention

The invention is defined in the claims.

The present invention relates to a method for assessing a subject's risk of having a cardiovascular disease comprising the step of measuring the level of inhibition factor 1 (IF1) in a body fluid sample obtained from said subject in which the IF1 level is negatively correlated with the risk of said subject of having cardiovascular disease.

A high level of IF1 is predictive of a low risk of having cardiovascular disease.

A low level of IF1 is predictive of a high risk of having cardiovascular disease.

The present invention also relates to a method for measuring the level of IF1 in a subject comprising the step of measuring the level of IF1 in a blood sample or in a sample of cerebrospinal fluid obtained from said subject.

The present invention provides a kit comprising means for measuring the level of IF1 in a sample of body fluid.

The invention also relates to an agent that increases the level of HDL selected from the group consisting of low lipid apoA-I, apoA-I associated with a mixture of phospholipids, apoA-I mimetics and CETP 35 inhibitors for use. in the treatment of cardiovascular disease in a subject that has a low level of IF1 in the blood.

Detailed description of the invention

Definitions

Cardiovascular disease (CVD) is the general term for diseases of the heart and blood vessels, including atherosclerosis, coronary heart disease, cerebrovascular disease, aortoiliac disease, and peripheral vascular disease. Subjects with CVD may develop numerous complications, including,

45 but without limitations, myocardial infarction, stroke, angina pectoris, transient ischemic attacks, congestive heart failure, aortic aneurysm and death.

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine and a primate. Preferably, a subject according to the invention is a human being.

The term "IF1" should be broadly understood, encompasses mature IF1, its isoforms and its fragments.

IF1 (inhibition factor 1) regulates the ATPase activity of mitochondrial F1F0-ATP synthase (Pullman ME et al. (1963)). IF1 is a 10 kDa basic mitochondrial protein encoded by a nuclear gene with a significant degree

55 homology between several species (Green DWet al. (2000)). IF1 joins through its N-end region at the interface between the α and β subunits of the F1 catalytic sector of mitochondrial ATP synthase, thereby blocking rotary catalysis (Cabezon E et al., (2003)) . It has been reported that exogenous IF1 could specifically bind ATP synthase of the cell surface (also called ecto-F1-ATPase) on hepatocytes and endothelial cells and reduces its hydrolytic activity (Martinez LO et al. (2003); Mangiullo R et al., (2008); (Burwick NR et al., (2005)). Examples of documents that disclose IF1 are Lebowitz et al. (1993); Jackson et al., (1988); Martinez et al. (2003 ); Mimura et al., (1993); Solaini et al., (1997) and W098 / 33909.

Recent reports have described the presence of IF1 in the plasma membrane of different cell types such as hepatocytes and endothelial cells (Burwick NR et al., (2005); Cortes-Hernandez P et al. (2005); Contessi 65 S et al . (2007); Giorgio V et al. (2010)).

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Alternative splicing occurs at the IF1 locus and three variants of the transcript encoding different isoforms of IF1 have been identified in humans (provided in Ref Seq http://www.ncbi.nlm.nih.gov/RefSeq/) . All precursor isoforms contain an identical mitochondrial signal peptide (or transit peptide) of 25 amino acids. Mature and functional isoforms of IF1 do not contain this transit peptide. Variant 1 of IF1

5 encodes the longest isoform, IF1 isoform 1, which consists of a 106 amino acid precursor protein and a mature 81 amino acid protein (ATIF1_HUMAN; REFSEQ: registration number NM_016311.3, Swiss-Prot registration number: Q9UII2 (Ichikawa N et al .; (1999)).

Variant 2 of IF1 encodes IF1 isoform 2 consisting of a precursor protein of 71 amino acids and a mature protein of 46 amino acids (A6NE74_HUMAN; REFSEQ: registration number NM_178190.1). Isoform 2 has a different and shorter C-terminus, compared to isoform 1, due to an alternative splice site in the 3 ’coding region and a phase shift.

Variant 3 of IF1 encodes the shortest isoform, IF1 isoform 3, which consists of a precursor protein of 60

15 amino acids and a mature protein of 35 amino acids (REFSEQ: registration number NM_178191.1). Variant 3 is identical to variant 1, but without the last 46 amino acids of the C-terminus.

In a preferred embodiment, IF1 is IF1 isoform 1.

In another embodiment, IF1 is IF1 isoform 2.

In another embodiment, IF1 is IF1 isoform 3.

Testing method

The present invention relates to a method for measuring the level of IF1 in a subject comprising the step of measuring the level of IF1 in a blood sample or in a sample of cerebrospinal fluid obtained from said subject.

In fact, the inventors have surprisingly demonstrated that IF1, known until now that it was present in mitochondria or in the plasma membrane, is a circulating protein present in the blood.

In one embodiment, the blood sample to be used in the methods according to the invention is a whole blood sample, a serum sample, or a plasma sample. In a preferred embodiment, the blood sample is a serum sample.

In another embodiment, the level of IF1 is measured in a sample of cerebrospinal fluid.

In fact, ATP synthase inhibited by IF1 is present in the plasma membrane of some brain cells. In particular, ATP synthase has been discovered in the superficial cell of hippocampal neurons and cultured astrocytes (Schmidt C et al., (2008)).

Once the subject's body fluid sample is prepared, the level of IF1 can be measured by any method known in the art.

For example, the concentration of IF1 can be measured using conventional electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction or sandwich-type assays. Such tests include, but are not limited to, Western blotting, agglutination assays, enzyme-labeled immunoassays and mid-immunoassays such as ELISA, biotin / avidin-type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, high performance liquid chromatography (HPLC), chromatography. size exclusion, solid phase affinity, etc.

In a particular embodiment, said methods comprise contacting the body fluid sample with a ligand.

As used herein, "ligand" refers to a molecule capable of selectively interacting with IF 1.

The ligand can generally be an antibody that can be polyclonal or monoclonal, preferably monoclonal. Polyclonal antibodies directed against IF1 can be sensitized according to known methods by administering the appropriate antigen or epitope to a host animal selected, for example, from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Several adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in the practice of the invention may be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against IF1 can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Production and isolation techniques include, but are not limited to, the hybridoma technique originally described by Kohler et al. Nature 1975; 256 (5517): 495-7; the human B lymphocyte hybridoma technique (Cote et al Proc Natl Acad Sci USA. 1983; 80 (7): 2026-30); and the EBV hybridoma technique (Cole et al., 1985, in "Monoclonal Antibodies And Cancer Therapy", Alan R. Liss, Inc. pp. 77-96). Alternatively, the techniques described for the production of single chain antibodies (see, for example, U.S. Patent 4,946,778) can be adapted to produce single chain antibodies directed against IF1. Antibodies useful in the practice of the present invention also include those directed against IF1, which include but are not limited to F (ab ') 2 fragments, which can be generated by pepsin digestion of the antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F (ab ') 2 fragments. Alternatively, Fab or scFv expression libraries can be constructed to allow rapid identification of fragments that have the desired specificity against IF1. For example, phage antibody expression libraries can be used. In such a procedure, single chain Fab fragments (scFv) or Fab fragments are expressed on the surface of a suitable bacteriophage, for example, M13. In summary, spleen cells of a suitable host, for example, mouse, which has been immunized with a protein, are removed. The coding regions of VL chains

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15 and VH are obtained from said cells that produce the desired antibody against the protein. Next, these coding regions are fused with one end of the phage sequence. Once the phage is inserted into a suitable carrier, for example, bacteria, the phage expresses the antibody fragments. The expression of phage antibodies can also be provided by combinatorial methods known to those skilled in the art. Antibody fragments expressed in a phage can then be used as part of an immunoassay.

In another embodiment, the ligand can be an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in terms of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the ability to recognize virtually any class of target molecules

25 with high affinity and specificity. Such ligands can be isolated by systematic evolution of ligands by exponential enrichment (SELEX) from a library of random sequences, as described in Tuerk et al. (1990) Science, 249, 505-510. The random sequence library can be obtained by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, optionally chemically modified, of a unique sequence. The possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena 1999. Peptide aptamers consist of variable regions of conformationally restricted antibodies expressed on a protein platform, such as E. coli thioxirredoxin A, which is selected from combinatorial libraries using two hybrid methods (Colas et al. (1996) Nature, 380, 548-50).

The ligands of the invention, such as antibodies or aptamers, can be labeled with a detectable molecule or substance, such as a fluorescent molecule, a radioactive molecule or other labels known in the art. Labels that generally provide a signal (both directly and indirectly) are known in the art.

As used herein, the term "labeled", with respect to the ligand, is intended to encompass the direct labeling of the antibody or aptamer by coupling (i.e., physical bonding) of a detectable substance, such as a radioactive agent. or a fluorophore (for example, fluorescein isothiocyanate (FITC) or phycoerythrin (PE) or indocyanine (Cy5)) with the antibody or aptamer, as well as an indirect labeling of the probe or antibody by reactivity with a detectable substance. An antibody or aptamer of the invention may be

45 labeled with a radioactive molecule by some method known in the art. For example, radioactive molecules include, but are not limited to radioactive atoms for scintigraphy studies such as I123, I124, In111, Re186, Re188.

The aforementioned assays generally involve the binding of the ligand (ie, the antibody or aptamer) to a solid support. The solid supports to be used in the practice of the invention include substrates such as nitrocellulose (for example, in the form of a membrane or microtiter well); polyvinyl chloride (for example, microtiter sheets or wells); polystyrene latex (eg, beads or microtiter plates); poly (vinylidene fluoride); diazoted paper; nylon membranes; activated beads, magnetically sensitive beads, and the like. More particularly, an ELISA method can be used, in which the wells

55 of a microtiter plate are coated with a set of antibodies directed against IF1.

A sample of body fluid containing or suspected of containing IF1 is then added to the coated wells. After a sufficient incubation period to allow the formation of ligand-IF1 complexes, the one or more plates can be washed to remove unbound material and a labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any marker protein captured in the sample, the plate is washed, and the presence of the secondary binding molecule is detected using methods well known in the art.

As a ligand, the secondary binding molecule may be labeled. 65

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Different immunoassays, such as radioimmunoassay or ELISA, have been described in the art.

Measurement of IF1 concentration with or without immunoassay-based methods may also include protein separation: centrifugation based on the molecular weight of the protein; electrophoresis based on the

5 mass and load; HPLC based on hydrophobicity; molecular exclusion chromatography depending on size; any solid phase affinity based on the affinity of the protein for the specific solid phase used. Once separated, IF1 can be identified based on a known "separation profile", for example, retention time, for said protein that is measured using conventional techniques. Alternatively, the separated proteins can be detected and measured by, for example, a mass spectrometer.

The concentration of IF1 can also be determined by measuring the activity of F1-ATPase using an ATP regeneration system described above (van Raaij MJ et al., (1996)). A unit of IF1 is defined as the amount that inhibits 0.2 U of ATPase by 50%, where 1 U of ATPase hydrolyzes 1 µmol of ATP / min. IF1 activity can be analyzed to determine the ATPase inhibition of both submitochondrial particles.

15 bovine animals that have been deprived of endogenous IF1, or isolated F1-ATPase, without nucleotides and free of inhibitors, (van Raaij MJ et al., (1996)).

In a preferred embodiment, the method for measuring the level of IF1 comprises the step of contacting the cerebrospinal fluid sample with a ligand capable of selectively interacting with IF1 to allow the formation of a ligand-IF1 complex.

In a more preferred embodiment, the method according to the invention further comprises the steps of separating any unbound material from the blood sample or the cerebrospinal fluid sample from the ligand-IF1 complex, contacting the ligand-IF1 complex with a labeled secondary binding molecule, separate

25 any unbound secondary binding molecule of the secondary binding molecule-IF1 complexes and measuring the level of the secondary binding molecule in the secondary binding molecule complexes-IF1.

The invention also provides the use of a kit comprising means for measuring the level of IF1 in a sample of body fluid to carry out the methods according to the invention.

The kit may comprise a solid support, a ligand capable of selectively interacting with IF1 that is bound to said solid support, and an assay to measure the level of the ligand-IF1 complex.

In a preferred embodiment, the ligand of the invention is an antibody that binds to the circulating IF1.

35 As used herein, "circulating IF1" refers to IF1 that is found in a body fluid of a subject, in particular, within the bloodstream.

Some antibodies against IF1 do not bind to circulating IF1. For example, the commercially available Invitrogen anti-IF1 of clone ID 5ED7 does not bind the circulating F1.

To analyze whether an antibody directed against IF1 binds to the circulating IF1, the antibody directed against IF1 must be analyzed by immunoprecipitation, as described, for example, in Plosone, September 2011, of Genoux A. et al.

For example, immunoprecipitation of the circulating IF1 can be carried out with the Pierce H Crosslink immunoprecipitation kit (Thermo Scientific) according to the manufacturer's instructions. 10 mg of antibody directed against IF1 to be analyzed are covalently crosslinked onto protein A / G resin and then incubated overnight with 2 mg of 500 mg of proteins from a serum rich in low-abundant proteins. 2 mg of recombinant IF1 (rIF1) or 500 mg of HepG2 cell proteins after cell lysis can be used positive control.

After washing, the bound proteins are eluted in 0.2 M glycine pH 2.8 and 56 Laemmli sample buffer is added. The presence of IF1 is controlled in the eluted fractions.

In a preferred embodiment, the kit is for an ELISA.

Diagnostic Method:

The invention also provides a method for assessing a subject's risk of having a cardiovascular disease comprising the step of measuring the level of IF1 in a sample of body fluid obtained from said subject in which the level of IF1 is negatively correlated with the risk of said subject of having a cardiovascular disease.

In fact, in addition to having discovered that IF1 is in circulation, the inventors have also shown that the level of IF1 in circulation is also negatively correlated with the risk of CVD.

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A high level of IF1 is predictive of a low risk of having cardiovascular disease.

A low level of IF1 is predictive of a high risk of having cardiovascular disease.

In a preferred embodiment, the body fluid sample is a blood sample (for example, a whole blood sample, a serum sample, or a plasma sample).

Typically, a high or low level of IF1 is obtained by comparison with a median control value or by comparison, respectively, with a higher or lower control value.

Said control values can be determined with respect to the level of IF1 present in samples taken from one or more healthy subjects or from the distribution of IF1 in a control population.

In one embodiment, the method according to the present invention comprises the step of comparing said level of

15 IF1 with a higher and / or lower control value in which a level of IF1 above said higher control value is predictive of a low risk of having cardiovascular disease and a level of IF1 below said control value Lower is predictive of a high risk of having cardiovascular disease.

Control values may depend on several parameters such as the methods used to measure the level of IF1 or the sex of the subject.

Typically, for a level of IF1 in a serum sample measured using a competitive immunoassay with a polyclonal antibody sensitized against human IF1, a level of IF1 greater than 0.65 µg / ml, at 0.7 µg / ml, at 0, 75 µg / ml, at 0.8 µg / ml or 0.85 µg / ml is predictive of a low risk of having cardiovascular disease, and a

IF1 level below 0.45 µg / ml, at 0.4 µg / ml, at 0.35 µg / ml, at 0.3 µg / ml, at 0.25 µg / ml is predictive of high risk of having a cardiovascular disease.

This correlation between the level of IF1 and the risk of having a CVD is independent of environmental markers of CVD risk such as physical activity, smoking, etc ...

Therefore, IF1 is a new and effective marker of CVD risk.

In one embodiment, it is not known, on the other hand, that the subject is at high risk of having cardiovascular disease.

The method according to the invention can also be combined with other methods to assess the risk of having cardiovascular disease. Examples of such methods are well known in the art.

Classic methods for assessing risk factors include, but are not limited to, evaluation of personal and / or family medical history of CVD, tobacco use, blood pressure measurements, plasma glucose, low density lipoprotein cholesterol (LDL ) and high density lipoprotein (HDL), cholesterol, triacylglycerides and the like.

In particular, IF1 levels will provide additional predictive value in those patients with low levels of HDL-cholesterol and / or apolipoprotein A-I.

The present invention also relates to a method for assessing the severity of a cardiovascular disease in a subject having said cardiovascular disease comprising the step of measuring the level of IF1 in a sample of body fluid obtained from said subject in which the level of IF1 is negatively correlated with the severity of cardiovascular disease in said subject.

In fact, the inventors have discovered that in a bivariate analysis, the average Gensini score, which reflects the severity of coronary artery disease, decreases regularly as the plasma concentrations of IF1 are higher.

55 Gensine's score is one of the most commonly used trials to estimate the severity of coronary artery disease (CA), as defined above. Combine the number of stenosis observed, each receiving a score number according to the importance of the narrowing and a coefficient that reflects the location of the lesion in the coronary tree (Gensini GG. (1983)). These results were confirmed by angiography, another method to assess the severity of cardiovascular disease. Angiography objectifies the severity of coronary artery lesions. Angiograms are interpreted by two independent operators. Different scores can be calculated taking into account the number of stenosis, the degree of intraluminal narrowing and the geographical importance of the stenosis in the coronary tree.

The method for assessing the severity of a cardiovascular disease according to the invention could be combined with other well-known methods, such as the Gensini score or for each coronary stenosis, the degree of luminal narrowing and its geographical importance.

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The present invention also relates to a method of controlling the effect of a therapy to treat cardiovascular disease in a subject comprising the step of measuring the level of IF1 in a first sample of

5 body fluid obtained from said subject in t1 and measure the IF1 level in a second sample of fluid obtained from said subject in t2 in which when t1 is prior to therapy, t2 is during or after therapy, and when t1 is during therapy, t2 is later during therapy or after therapy, and in which an increase in the level of IF1 in the second sample compared to the level of IF1 in the first sample is indicative of a positive effect of Cardiovascular disease therapy of the treated subject.

In addition to current statin therapies for cardiovascular disease, HDL therapies have been developed. These therapies are intended to increase the level of HDL. In fact, as described above, there is an inverse correlation between HDL and the risk of CVD. Therefore, it has been reported that increasing HDL 1 mg / dL reduces the risk of CVD by 2 or 3% (Gordon DJ, Rifkind BM. High-density lipoprotein: the clinical implications

15 of recent studies. N Engl J Med. 1989; 321: 1311-1316; Castelli WP, Anderson K, Wilson PW, Levy D. Lipids and risk of coronary heart disease: The Framingham Study. Ann Epidemiol. 1992; 2: 23-28).

There are different therapeutic approaches to HDL therapy. Many approaches are aimed at increasing the level of HDL or apolipoprotein A-I (apoA-I), the main atheroprotective apolipoprotein of HDL. It could be expected that parenteral infusion of apoA-I or apoA-I with low lipid content of full length associated with a phospholipid mixture would have beneficial effects so that, with repeated infusions, it could have a favorable impact on atherosclerosis (Nissen SE et al., (2003)). Selective delipulation of ex vivo HDL, which generates lipid-poor apoA-I is an alternative strategy; In this model, the slipped HDL is re-infused into the patient, which would decrease the development of atherosclerosis (Waksman R et al., (2010)). The mimetics of ApoA-I

25 are other therapeutic approaches for HDL therapy. These apoA-I mimetics are small amphipathic peptides of 18-22 amino acids based to some extent on the apoA-I sequence that has properties similar to apoA-I, including the abilities to promote the removal of excess cholesterol from tissues. (Navab M et al., (2005)). An important advantage of these mimetic apoA-I peptides over full-length apoA-I is that it is cheaper and easier to manufacture as a therapeutic molecule.

Some ApoA-I mimetics have been identified; these include ETC-216 from Esperion, D-4F from Kos Pharmaceuticals, AVP-26452 from Astrazeneca. Examples of these drugs are disclosed in Navab M et al. (2005); Garber DW et al. (2001); Navab M et al. (2004); Navab M et al. (2006) and document WO2010093918.

35 Most known drugs that inhibit the degradation of HDL are CETP inhibitors. The term "CTP inhibitors" refers to compounds that inhibit cholesterol-mediated transport of the cholesteryl ester transfer protein (CETP) of different cholesteryl esters and triglycerides from HDL to LDL and VLDL.

CETP inhibitors are well known to those skilled in the art. Examples of patents disclosing CETP inhibitors are WO2009071509, WO2009059943, WO2008082567, EP2007726, EP1973889, US 6,140,343, US 6,197,786, 6,723,752, WO 2006/014357; WO2006 / 014413, WO2007 / 079186, EP1670765 or US 5,512,548.

Several effective CETP chemical inhibitors have been identified; these include torcetrapib (Pfizer, New York, NY,

45 USA), dalcetrapib (formerly known as RO4607381 / JTT-705, Roche / Japan Tobacco, Basel, Switzerland), and anacetrapib (MK-0859, Merck & Co., Whitehouse Station, NJ, USA) . A molecular look at the mechanism of action of these inhibitors has been possible as a result of the crystal structure of the CETP (see for example, et al., European Heart Journal (2010) 31, 149-164 for review).

The invention also relates to an agent that increases the level of HDL selected from the group consisting of low lipid apoA-I, apoA-I associated with a mixture of phospholipids, apoA-I mimetics and CETP inhibitors for use in the treatment of a cardiovascular disease in a subject that has a low level of IF1 in the blood.

Typically, a sample of body fluid from the subject is obtained and the level of IF1 is measured in this sample, together with the evaluation of plasma lipids, HDL and LDL-cholesterol and / or apolipoproteins AI and B. Of course, statistical analyzes revealed that the increase in IF1 levels would be especially beneficial in those patients presenting with low levels of HDL-cholesterol and / or apolipoprotein AI.

In a preferred embodiment, the cardiovascular disease is a coronary artery disease.

In a preferred embodiment, the cardiovascular disease is atherosclerosis.

The invention also relates to the use of IF1 as a marker of cardiovascular risk.

The invention will be further illustrated by the following figures and examples.

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Description of the figures

Figure 1: Typical standard curve used for quantification of IF1.

5 Figure 2: IF1 frequency distribution among the general population. Density histogram, with the normal density graph of IF1. IF1 was measured in 669 controls aged between 45-74. Figure 3: Relative risk of CVD as a function of IF1. Figure 4: Relative risk of CVD based on IF1 after adjustment according to education, physical activity, smoking and alcohol habits, diabetes, dyslipidemia, hypertension, levels of C-reactive protein (CRP) and apoA-I. Figure 5: Severity of coronary lesions as a function of IF1 - Gensini score Figure 6: Severity of coronary lesions as a function of IF1 - left ventricular ejection fraction (%)

Example

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Materials and methods

Sample treatment for analysis by mass spectrometry

100 µl of serum from 10 individuals selected from the general population of the GENES cohort were combined with each other. The GENES study is an investigation of the genetic and environmental determinants of coronary artery disease carried out within the framework of a large sectional case control at Toulouse University Hospital.

This combination of plasmas was then prefracted with the ProteoMiner ™ kit (Bio-Rad Laboratories) from

25 in accordance with the manufacturer's protocol. The protein concentration in the sample was 2.5 g / l. The equalized plasma sample was stored at -80 ° C until mass spectrometry analysis.

The total ProteoMiner ™ elution volume of the plasma sample was diluted in Laemmli sample buffer. The reduction and alkylation of the cysteines was carried out by 1 h incubation at room temperature (20 mM DTT was included in the Laemmli sample buffer) followed by the addition of 90 mM iodoacetamide for 30 min at room temperature in the dark. Next, the sample was loaded into four rows of a 15% SDS-PAGE acrylamide gel and another four rows were loaded into the same gel with 5 µg of recombinant human IF1, rIF1. The proteins were visualized by Coomassie blue staining and four visible bands corresponding to rIF1 were cut. In parallel, a portal with the same molecular size of rIF1 was cut to

35 each of the four rows of palm samples. The gel slides were washed with three washings with 100 mM ammonium bicarbonate for 15 min at 37 ° C followed by a second wash in 100 mM ammonium bicarbonate, acetonitrile (1: 1) for 15 min at 37 ° C. The proteins were digested by incubating each gel carrier with 0.6 µg of modified sequencing quality trypsin (Promega) in 50 mM ammonium bicarbonate overnight at 37 ° C. The resulting peptides were extracted from the gel by three steps that were combined together: a first incubation in 50 mM ammonium bicarbonate for 15 min at 37 ° C and two incubations in 10% formic acid, acetonitrile (1: 1) for 15 min at 37 ° C, and combined with each other. The four peptide extracts corresponding to the same initial sample (rIF1 or plasma sample) were combined and dried in a SpeedVac, and resuspended in 5% acetonitrile, 0.05% trifluoroacetic acid.

45 Targeted analysis by monitoring multiple reactions

The mixture of rIF1 peptides was used for optimization of the detection of human IF1 by nanoCL / MS in multiple reaction monitoring mode (MRM) using an Ultimate3000 system (Dionex, Amsterdam, The Netherlands) coupled with a 5500 QTrap mass spectrometer ( AB Sciex, Foster City, CA, USA). 1 pmol of rIF1 was loaded into a C18 pre-column (300 µm internal diameter x 5 mm, Dionex, Amsterdam, The Netherlands) at 20 µl / min in 2% acetonitrile, 0.05% trifluoroacetic acid. After 3 min of desalting, the pre-column was changed in line by the analytical column (75 µm internal diameter x 15 cm of silica capillary tube packed with Reprosil-Pur C18-AQ 3 µm phase, Dr. Maisch, GmbH, Germany) balanced in 100% solvent A (5% acetonitrile, 0.2% formic acid). The peptides were eluted using a gradient 0-50% solvent B (80% acetonitrile, 55 0.2% formic acid) for 25 min at a flow rate of 300 nl / min. The 5500 QTrap worked in MRM mode together with the Analyst software (version 1.5.1, AB Sciex, Foster City, CA, USA). The four human IF1 peptides that provided the strongest signals were monitored in MRM with quadrupoles Q1 and Q3 configured in unit resolution, optimal collision energies and cluster breakage potentials were determined for three parent / fragment transitions for each peptide . During the analysis, EMEM complete scanning (EPI) enhancements were also initiated when the MRM signal exceeded 200 counts, with a mass tolerance of 250 mDa, the linear ion trap (LIT) was set to a 150 ms fixed filling time. A third of the peptide extract from the plasma sample was then loaded into the system analogously to rIF1, and the analysis with the 5500QTrap was carried out in the same way (MRM + EPI) with the optimized transition list. A blank analysis was carried out under the same conditions before analyzing the

65 plasma sample to ensure non-contamination of the nanoCL system by rIF1.

image9

Search in the database

The Mascot Daemon software (version 2.3.2, Matrix Science, London, UK) was used to perform the database searches. To automatically extract peak lists from Analyst wiff files, the

5 macro Mascot.dll (version 1.6b27) provided by Analyst and configured for non-clustering of EM / EM scans. Homo sapiens input data were searched in the Uniprot protein database (September 2010 version - 96635 sequences). The carbamidomethylation of the cysteines was configured as a fixed modification, and the oxidation of the methionines and the deamidation of the asparagine and glutamines was configured as the variable modifications for Mascot searches. The specificity of trypsin digestion was established for excision after Lys or Arg except before Pro, and the lack of two trypsin excision sites was allowed. Mass tolerances in MS and MS / MS were set to 0.6 Da, and the instrument configuration was specified as "ESI-QUAD".

Construction of the bacterial expression plasmid.

The sequence encoding the mature peptide of human IF1, without the sequence of the mitochondria-directed signal (MTS), was amplified by PCR from cDNA from HeLa cells. The amplified fragment was then introduced into BamHI / HindIII restriction sites in the VariFlex pBEn-SBP-SET1-Qa (Stratagene) expression vector containing the streptavidin-binding peptide coding sequence (SBP tag) and a site of cleavage of thrombin protease to allow the fusion of the SPB tag to the N-terminus of the coding sequence of the cloned protein. The resulting expression plasmid, which contains the complete mature human IF1, was verified by DNA sequencing and named as pBEn-SBP-IF1.

Excessive expression of human IF1 in Escherichia coli.

A colony of newly transformed E. coli BL21 (DE3) cells was inoculated into 30 ml of Terrific broth (Ozyme) containing 100 µg / ml ampicillin. The culture was grown overnight with shaking in a 1L flask at 37 ° C. Protein expression was induced by adding 150 ml of Terrific broth medium containing 100 mM IPTG, and growth continued for a further 3-4 h. The bacteria were centrifuged at (7000 g, 15 min, 4 ° C) and the bacterial agglomerate was kept at -20 ° C.

Purification of human IF1 expressed in bacterial form.

All procedures were carried out at 4 ° C. The cell agglomerate that contained the IF1 protein

Recombinant was resuspended in 20 ml of lysis buffer (0.5 M NaCl, 0.7 M sucrose, 1 mM EDTA, 1% Triton X100, 5 mM β-mercaptoethanol, 1x Roche protease inhibitor cocktail) then it was sonicated 3 times for 100 s. The recombinant proteins were soluble in the cytoplasmic fraction that was clarified by high speed centrifugation (130,000 g, 1 h, 4 ° C). The broken cell supernatant containing IF1 was diluted in binding buffer (50 mM ammonium carbonate, 0.5 M NaCl, pH 10.0) to a final concentration of 1 mg / ml and filtered through a filter 0.22 µm and then applied at 1 ml / min to a HiTrap ™ Streptavidin HP 1 ml column (GE Healthcare) equilibrated with binding buffer. The column was washed until the optical density at 280 nm reached 0.1, then IF1 was evaluated from the column in 6 M guanidine elution buffers. The combined fractions containing the IF1 protein fused with SBP tag were dialyzed. times against thrombin cleavage buffer (20 mM Tris-HCl pH 8.4, 150 mM NaCl, 2.5 mM CaCl2) and thrombin (10 U / mg protein) was added

45 overnight at TA to split the SPB tag. Next, the sample was loaded on a 1 ml HiTrap ™ Streptavidin HP column and pure IF1 was found in the piston flow fraction. Human recombinant IF1 was> 95% pure as determined by SDS-PAGE (data not shown).

Preparation of IF1 antiserum

A polyclonal antibody was sensitized in New Zealand White rabbits against human recombinant IF1. Total serum IgG was purified from serum by affinity chromatography with the use of Sepharose CL-4B protein columns (GE healthcare), according to the manufacturer's instructions. The purity of the IgG fractions were evaluated by sodium polyacrylamide dodecyl sulfate gel electrophoresis (SDS-PAGE). IgG

Specific against IF1 was purified from total serum IgG by affinity chromatography using the IF1 column.

Immunofluorescence

HeLa cells cultured on coverslips were incubated with Mitotracker for 30 min at 37 ° C when indicated. Subsequently, the cells were washed, fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 60 min. After saturation of the non-specific sites with PBS containing 10% FCS, the cells were first incubated with the primary antibody (monoclonal antibody directed against Invitrogen alpha ATP synthase; polyclonal antibody directed against IF1) then with labeled secondary antibodies

65 fluorescently from Jackson Immunoresearch Laboratories (West Grove, PA). The images were taken with a Zeiss LSM 510 confocal confocal microscope with a 63-magnification Plan-Apochromat lens.

image10

SPR analysis (Biacore).

The antibody directed against purified IF1 was immobilized by amine binding on CM5 chips (Biacore AB) after activation of NHS-EDC. Binding was analyzed in a Biacore 3000 apparatus. Increasing concentrations of IF1

5 human recombinants were injected at a flow rate of 20 µl / min, exposed to the surface for 175 s (association phase), followed by 200 s of circulating flow rate during which dissociation occurred. Between injections, to recover the initial level prior to binding, the surface of the detector chip was regenerated by a 5 µl injection of 0.01% SDS (15 s contact time). The apparent Kd value was calculated using the CLAMP software (Myszka DG, Morton TA, CLAMP: a binensor kinetic data analysis program, Trends Biochem Sci. April 1998; 23 (4): 149-50).

IF1 immunoassay

A competitive assay was designed to quantify IF1 in human serum. Wells of Polysorb ELISA 96 plates

15 wells (NUNC, Roskilde, Denmark) were coated with 100 µl of recombinant human IF1 (0.5 µg / ml) diluted in bicarbonate buffer (0.1 M, pH = 9.6). Plates were incubated overnight at room temperature (RT) to allow complete binding. Serum samples were thawed at room temperature and centrifuged (2,000 x g, 10 min). 50 µl of serum from each subject (diluted with 50 µl of 1X PBS), or 100 µl of each standard human recombinant IF1 (0; 0.025; 0.050; 0.075; 0.1; 0.3; 1 and 2.5 µg / ml), were incubated with 100 µl of polyclonal antibody directed against biotinylated IF1 (dilution: 1/1000 in PBS-Tween buffer 0.05% -1% BSA) overnight at 4 ° C. The plates were then washed 3 times with PBS buffer pH 7.4 / 0.05% Tween 20 and incubated with 200 µl per well of blocking buffer (PBS buffer pH 7.4% / 3% BSA) for 1 hour at TA. After washing with PBS 3 times, the sample / antibody mixtures were added to the wells and incubated for 4 hours at RT. The plates were washed again and incubated with 100 µl per well of streptavidin-HRP (dilution: 1/5000 in

25 PBS buffer pH 7.4% -Tween 0.05% -1% BSA) (Invitrogen, Cergy Pontoise, France) for 1 hour at RT. After washing 3 times with 0.05% PBS-Tween buffer, the plates were incubated with 200 µl per well of TMB substrate (3,3 ', 5,5'-tetramethylbenzidine) with horseradish peroxidase (HRP) for 20 minutes to TA. The reaction was stopped with 50 µl per well of 1 N HCl, and the plates were then read at 450 nm in a microplate reader (Varioscan Flash, Thermo electron corporation). The optical density was subtracted at 570 nm (background). Sample concentrations were determined using the adjustment of the standard curve (type of adjustment: 4-parameter logistic). Figure 1 shows the typical standard curve, in the range of 0.025 to 2.5 µg of IF1 / ml. A fifty percent displacement was obtained for a concentration of 0.4 µg / ml. Repeatability (same day) and reproducibility (10 measurements over a period of 6 months) provided coefficients of variation of 6-7%. In the dilution experiments an average recovery of 96.8% was measured. Resistance to freezing and thawing was evaluated

35 on different occasions, with a concordance of 95% -103% between measurements. With respect to the possible correlations of IF1 with HDL markers, competition experiments with purified apolipoproteins A-I and A-II indicated the absence of cross-reactivity in this immunoassay.

CVD subjects and control subjects

Serum IF1 was evaluated both in the general population and in subjects with cardiovascular disease (CVD). The evaluation was carried out in the context of GENES. A collection of biological samples had been established (declared as DC-2008-403 No. 1 in the Ministry of Research and the Regional Health Agency).

45 Cases were subjects admitted for a first coronary episode, both unstable angina pectoris or defined myocardial infarction. All cases were evaluated according to a strict protocol, which included clinical, electrocardiographic, biological and angiographic criteria. They were explored for the evaluation of risk factors after ≥ 3 months of waiting after the CVD event. Controls were randomly selected from the general population, using electoral censuses. Cases and control subjects were matched by age. Overall, the cohort included 791 male subjects with CVD and 817 age-matched controls, aged 45-75. Data on eating habits, physical activity, tobacco and alcohol consumption, educational level were collected. The presence of dyslipidemia, diabetes or hypertension was evaluated both from the current treatments of the subjects

or of the clinical and biological evaluation of the subject during the investigation. The clinical investigation included anthropometric variables, blood pressure and heart rate, and for cases of CVD, a cardiological evaluation

55 complete. Biological measurements focused on lipids and lipoproteins, glucose and insulin, gamma GT and sensitive CRP. Serum IF1 was measured in the first 669 controls and 648 cases.

Results

Characterization of IF1 in human plasma

The presence of IF1 in human serum was demonstrated unambiguously by mass spectroscopy analysis.

65

image11

Characterization of the antibody directed against IF1

The interaction of human recombinant IF1 (rIF1) with the polyclonal antibody directed against IF1 was monitored by surface plasmon resonance analysis (SPR). Under these conditions, rIF1 specifically bound the antibody directed against IF1 immobilized on SPR chips (KD = 6.1 nM). To further assess the specificity of the antibody directed against IF1, immunofluorescence experiments were carried out on HeLa cells. Immunolabeling using the antibody directed against IF1 showed a characteristic mitochondrial pattern. This was further confirmed by simultaneous localization of IF1 staining with mitochondrial markers such as MitoTracker or the alpha subunit of ATP synthase. These data indicate that the

10 antibody directed against IF1 was able to specifically recognize endogenous mitochondrial IF1.

Serum IF1 levels in the general population

The graphical representation of IF1 levels in the general male population showed a normal distribution, 15 with a median value of 0.53 µg / ml and a 95% confidence interval of 0.24-0.82 µg / ml. (Fig. 2)

Correlation with plasma lipid parameters

The correlation between IF1 levels and plasma lipid levels was investigated. The plasma IF1 is

20 positively correlated with HDL cholesterol, apo AI and levels of lipoparticle AI, and negatively with triglycerides, apolipoprotein B and lipoproteins containing apolipoprotein B. IF1, HDL-C and apo AI were negatively correlated with body mass index and waist circumference Several environmental factors, such as smoking, alcohol consumption and physical activity or an inflammatory condition documented by an elevation in CRP, showed the expected correlations with HDL-C or apo A-I. On the contrary, the

25 correlations of these factors with IF1 were bad or absent (Table I). This indicates that the correlations between IF1 and the HDL markers are independent of the environmental variables.

Table 1: Correlation between IF1, apoA-I and HDL cholesterol and other risk factors in the general population (* p <0.05, ** p <0.01, *** p <0.001).

Parameters

Spearman interval correlation with IF1 (mg / l), p Spearman interval correlation with apoA-I (g / l), p Spearman interval correlation with HDL cholesterol (g / l), p

HDL cholesterol (g / l)

0.24, *** 0.82, *** one

ApoA-I (g / l)

0.27, *** one 0.82, ***

ApoB (g / l)

-0.17, *** -0.07, NS -0.15, ***

Triacylglycerides (g / l)

-0.25, *** -0.19, *** -0.48, ***

LpB: CIII (mg / l)

-0.14, *** -0.06, NS -0.34, ***

Lp B: E (mg / l)

-0.26, *** -0.21, *** -0.25, ***

Body Mass Index (BMI)

-0.17, *** -0.20, *** -0.33, ***

Systolic blood pressure (mmHg)

-0.02, NS 0.01, NS -0.08, *

Physical Activity Score

0.01, NS 0.09, * 0.20, ***

Alcohol (g / day)

0.09, * 0.28, *** 0.20, ***

Cigarettes (/ day)

-0.03, NS -0.10, * -0.10, *

CRP (mg / l)

-0.01, NS -0.13, *** -0.19, ***

30 This hypothesis was confirmed by multivariable analysis performed among control subjects. When apo A-I was considered as the dependent variable in statistical models that include IF1, known determinants were discovered, such as smoking, alcohol consumption, physical activity, BMI, inflammation status or presence of a patent dyslipidemia. However, IF1 was identified as a strong positive determinant for

35 concentrations of apoA-I, contributing to 26% of the total model (Table 2A). Table 2A shows the regression coefficients and their statistical significance in a multiple regression analysis. The calculated contribution of each parameter to the total statistical model is provided in the right column.

Table 2A: Determinants apo A-I in controls (multivariate analysis)

Parameters

β p

Dyslipidemia

-0.097 0.001

ApoB

-0.1011 0.02

Triglycerides (log)

-0.0439 0.03

Cigarettes / day

-0.0027 0.04

BMI

-0.0093 0.001

Alcohol

0.0021 0.001

CRP

0,1207 0.003

image12

Conversely, when serum IF1 was studied as the dependent variable, three important explanatory variables were identified: apoA-I (positive relationship), triglycerides and presence of dyslipidemia (negative associations) (Table 2B).

Table 2B: IF1 determinants in controls (multivariate analysis)

Parameters

β p

ApoA-I

1,116 0.001

Triglycerides (log)

-0.333 0.001

Dyslipidemia

-0,175 0.03

Together, these data support the idea that 1) IF1 is strongly associated with HDL markers, appearing as an independent determinant, 2) IF1 is negatively related to triglyceride-rich lipoprotein levels.

10

Serum levels of IF1 and cardiovascular disease.

The cases of CVD differed from the control subjects due to an increase in the prevalence of classic risk factors: hypertension, diabetes, dyslipidemia, higher BMI and tobacco consumption, lower physical activity. Too

15 showed higher levels of lipoproteins containing triglycerides and lower HDL markers (not shown). The average concentration of IF1 was 0.43 (± 0.13) in cases versus 0.53 (± 0.15) µg / ml in controls (p <0.001).

The probability index for CVD based on IF1 was first studied by sorting in

20 quartiles Compared to the first IF1 distribution quartile, quartiles 2, 3 and 4 showed a regular decline in CVD risk (Figure 3). As an alternative, IF1 was considered as a continuous variable, evidencing an inverse linear relationship between the concentrations of IF1 and the risk of CVD (not shown). In a multivariable model that includes classic risk factors, physical activity, smoking and alcohol habits, CRP and lipoprotein levels, IF1 remained negatively related to the risk of CVD, the quartile being associated

25 of higher IF1 with a 60% reduction in CVD events (Figure 4). This supports the hypothesis that IF1 could be a negative predictor independent of cardiovascular risk.

In addition, a statistical interaction appeared between the levels of IF1 and apo A-I or HDL-C (p <0.001). To explore this interaction, the concentrations of IF1 and apo A-I were divided into tertiles and the risk reduction of

30 CVD, taking as a reference, subjects who simultaneously had low levels of IF1 and apo A-I. After adjustment with a large panel of risk factors, intermediate and high levels of apo A-I transmitted a reduction in risk of> 90%, regardless of IF1 levels. For low concentrations of apo A-I, an increase in IF1 induced a gradual reduction in CVD risk. This indicates that a high IF1 can compensate for a low apo A-I in protection against cardiovascular events. Therefore, in subjects with low

35 levels of HDL, increased levels of IF1 could delay the catabolism of HDL particles, prolonging their atheroprotective effects.

Finally, the relationship between IF1 and the severity of CAD was studied, using several indices. From the angiographic data, the Gensini score was calculated, evaluating, for each coronary stenosis, the degree of

40 luminal narrowing and its geographical importance. As shown in Tables 5 and 6, the severity of CAD lesions gradually decreased as a function of IF1 levels. The left ventricular ejection fraction (LVEF) was analyzed by echocardiography. A positive correlation was observed with plasma IF1 concentrations. This last observation indicates that, among CAD subjects, myocardial function is less altered in patients with high plasma IF1 levels.

45 In conclusion, IF1 is a new independent determinant of apo A-I and HDL-cholesterol levels. In addition, an independent negative association between plasma IF1 and the onset of CVD and with the severity of the disease has been discovered. The plasma IF1 assessment will help document the cardiovascular risk profile, especially in those with low levels of apolipoprotein A-I and / or HDL-cholesterol.

fifty

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Claims (11)

image 1 1. A method for assessing a subject's risk of having a cardiovascular disease comprising the step of measuring the level of inhibition factor 1 (IF1) in a body fluid sample obtained from said subject in which 5 the level of IF1 is negatively correlated with the risk of said subject of having cardiovascular disease. 2. The method according to claim 1 comprising the step of comparing said IF1 level with a higher and / or lower control value in which:

-

a level of IF1 above said higher control value is predictive of a low risk of having cardiovascular disease and / or

-

a level of IF1 below that control value is predictive of a high risk of having a disease

cardiovascular. fifteen

3.

A method for assessing the severity of a cardiovascular disease in a subject having said cardiovascular disease comprising the step of measuring the level of IF1 in a sample of body fluid obtained from said subject in which the level of IF1 is negatively correlated with the severity of cardiovascular disease in said subject.

Four.

A method to monitor the efficacy of a therapy to treat cardiovascular disease in a subject comprising the stage of:

- measuring the level of IF1 in a first body fluid sample obtained from said subject in t1 and 25 - measuring the level of IF1 in a second body fluid sample obtained from said subject in t2 in which:

-

 when t1 is prior to therapy, t2 is during or after therapy, and

-

 when t1 is during therapy, t2 is later during therapy or after therapy,

in which an increase in the level of IF1 in the second sample compared to the level of IF1 in the first sample is indicative of a positive effect of therapy on the cardiovascular disease of the treated subject. The method according to any one of claims 1 to 4 wherein said body fluid sample is a blood sample.

6.

The method according to any one of claims 1 to 5 wherein said cardiovascular disease is a disease of the coronary arteries.

7.

The method according to any of claims 1 to 6 wherein said cardiovascular disease is atherosclerosis.

8. A method for measuring the level of IF1 in a subject comprising the step of measuring the level of IF1 in a blood sample or in a sample of cerebrospinal fluid obtained from said subject.

9.

The method according to claim 8 comprising the step of contacting the cerebrospinal fluid sample with a ligand capable of selectively interacting with IF1 to allow the formation of a ligand-IF1 complex.

10.

 The method according to claim 9 comprising the steps of:

- separate any unbound material from the blood sample or the cerebrospinal fluid sample from the ligand-IF1 complex, 55 - contacting the ligand-IF1 complex with a labeled secondary binding molecule, -separate any unbound secondary binding molecule from the secondary binding molecule complexes-IF1 and - detect the secondary binding molecule of the secondary binding molecule complexes-IF1. 11. An agent that increases the level of HDL selected from the group consisting of low lipid apoA-I, apoA-I associated with a mixture of phospholipids, apoA-I mimetics and CETP inhibitors for use in the treatment of a cardiovascular disease in a subject, in which said subject has been identified as having a low level of IF1 in the blood. 65 12. Use of a kit for carrying out the method according to any one of claims 1-10, said kit comprising: 16 image2 -a solid support, - a ligand capable of selectively interacting with IF1 that is bound to said solid support, and - an assay to measure the level of the ligand-IF1 complex, in which the ligand is an antibody that binds to the circulating IF1. 13. Use of IF1 as a marker of cardiovascular risk. 17